High-throughput sequencing has become one of the foundations for modern molecular biology, biotechnology, medicine and other fields. In recent years, studies on rapid, accurate and economic methods for determining gene expression level and nucleotide sequence have achieved continuous innovation; the second generation of high-throughput sequencing technology with sequencing by synthesis as the basic principle has become mature. The major sequencing companies have focused on the development of new sequencing products, shortening the process of sequencing and cost reduction. The currently existing sequencing products based on the second-generation sequencing technology include whole genome resequencing, whole transcriptome sequencing, and small-molecule RNA sequencing and the like. In particular, the application derived from the second-generation sequencing combined with microarray technology—target sequence capture sequencing technology can use a large number of oligonucleotide probes to complementarily bind to specific regions in the genome to capture and enrich gene fragments from the specific regions for sequencing; and for the detection, diagnosis and research of disease genes.
Complete Genomics (CG) Corporation currently has an independently developed second-generation sequencing technology suitable for human whole genome sequencing. The process for its library construction includes: genomic DNA disruption, the first linker ligation, double-strand cyclization and digestion, the second linker ligation, single-strand separation and cyclization, wherein the two linker ligations are very important in the process for library construction. The linker is a specially designed DNA sequence and when fixed to both ends of the DNA fragment by ligation or the like, can be identified during sequencing and used as a starting site for sequencing to enable the instrument to read the subsequent sequence information. In order to ensure that the read sequence information is easy to analyze, it is necessary to add two different linkers at both ends (5′ and 3′ ends) of a DNA fragment; in order to achieve this particular directional ligation, while avoiding the interconnection between the linkers, sticky end can be used to link the linkers; however, this requires fragments with sticky ends, which make it difficult to avoid the interconnection between fragments. The Complete Genomics Corporation uses multiple steps to add linkers to both ends respectively for construction of a sequencing library. In order to obtain a fragment with linkers at both ends, it is necessary to go through the following five steps: ligating a linker to one end of a DNA fragment; performing denaturation, annealing and extension; then ligating a linker to the other end of the DNA fragment; filling the vacancy; and performing a polymerase chain reaction. However, the multiple extension reactions therein require expensive reagents and multiple purification steps are required between the steps, thus resulting in high overall costs and lack of efficiency.
In order to solve the problems that too many steps are required for linker ligation in the construction of the sequencing platform library of Complete Genomics, the time for constructing the whole library is too long and the cost is too high, the present invention is proposed.